Cancer is a collective term for diseases caused by an uncontrolled division of cells in the body. Frequently the term refers specifically to the malignant growth or tumor resulting from such uncontrolled division of cells. Historical records show that cancer has scared and eluded humanity since ancient times. In the world of today, with better healthcare and increasing lifespans, the prevalence of cancer seems to have increased. While some forms of cancer can be cured, the outcome is far from certain, and cancer still remains an enigmatic and frightening diagnosis.
In his award winning book “The Emperor of All Maladies: A Biography of Cancer”, Siddhartha Mukherjee, Ph.D., M.D. states that “it is unclear whether an intervention that discriminates between malignant and normal growth is even possible. Perhaps cancer, the scrappy, fecund, invasive, adaptable twin to our own scrappy, fecund, invasive, adaptable cells and genes, is impossible to disconnect from our bodies. Perhaps cancer defines the inherent outer limit of our survival. As our cells divide and our bodies age, and as mutations accumulate inexorably upon mutations, cancer might well be the final terminus in our development as organism.”
The present inventor however disagrees with the above conclusion. Instead, the present inventor notes that there is a phase in the development of mammals when cell proliferation is extremely fast and efficient, but during which there are very few if any occurrences of cancer. This is the gestation period, i.e. the time in which a fetus develops, beginning with fertilization and ending with birth. The duration of this period varies between species. In humans, the gestation period is about nine months. Smaller, short lived mammals generally have a shorter gestation period, e.g. about 19 to 21 days for mice, and about 31 days for rabbits. Conversely, larger, long lived mammals tend to have a longer gestation period, e.g. about 21 months for elephants, and about 14 to 16 months for sperm whales.
During the gestation period, an extremely rapid cell differentiation and proliferation takes place. It appears that also others have noticed this, for example Vincent T. DeVita Jr. and Elizabeth DeVita-Raeburn in their book “The Death of Cancer: After Fifty Years on the Front Lines of Medicine, a Pioneering Oncologist Reveals Why the War on Cancer is Winnable—and How we can Get There” (Sarah Crichton Books, 2015) conclude that “embryonic cells have only nine months to make a baby: they must work very fast”. Nevertheless, very few “mistakes” happen during this fast work. Obviously there is either an absence of disturbing factors, or very effective control and repair mechanisms at work. The present inventor has accepted the challenge to identify these, and to put them to use in combatting cancer.
The present inventor is of course aware of the fact that there are instances where a fetus develops neuroblastoma, leukemia or teratoma, but these diseases are extremely rare. As the metabolism and development of the mammalian fetus is regulated by the same genes as in the adult mammal, it becomes interesting to determine what conditions or factors, molecular or other, differ between the period spent in utero, and the life post partum.
Additionally, the fast and well-regulated proliferation phase must then be quickly and adequately down-regulated. The similarities and differences between the well-regulated fast proliferation in the embryonal phase and the unregulated, pathological proliferation encountered in various forms of cancer have been the subject of extensive research. A number of relevant articles are listed in the attached list of references, incorporated herein by reference.
Understanding the factors regulating both benign and malignant cell proliferation is crucial for the capability to predict, detect and treat cancer. Important work is being performed in vitro, in cell culture. More specifically, the techniques of in vitro cell, tissue and organ culture is of great importance to pure and applied science, medicine and industry. Amongst others, it is considered a major replacement alternative in animal experimentation. This use poses high requirements on the reliability and repeatability of the experiments.
Cells require a complex environment in order to survive and proliferate in vitro. Therefore, in order to successfully culture cells in vitro, culture medium needs to be added to the cells. Usually, the culture medium contains animal serum as this contains basic components, such as hormones, growth factors, essential elements, proteins, amino acids, nucleic acids, etc. The most common type of serum used for cell growth is fetal bovine serum (FBS), also known as fetal calf serum (FCS). Serum from other species is also available.
FBS is obtained from fetuses harvested in abattoirs from healthy dams fit for human consumption. Sera is also available from other species, such as horse, cat, dog, rabbit, et cetera, and used primarily when cells from said species are cultured. In the cell culture, the serum provides a source of a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water-insoluble components, and other compounds necessary for in vitro growth of cells, such as hormones and attachment factors. Serum also adds buffering capacity to the medium and binds or neutralizes toxic components. So far, attempts to replace serum entirely with serum-free medium have met only with limited success. Serum must therefore be considered to be a raw material or reagent of crucial importance for research and manufacture within the entire life science sector.
In the serum manufacturing process, whole blood is aseptically collected and allowed to clot. Once the serum has been separated from the clot, it is pooled and frozen. Selected batches are then thawed, tested for endotoxins and hemoglobin content and only the accepted material is pooled and thoroughly blended. The pooled sera is then sterile filtered using a sequence of pre-filters and membrane filters. For example a 0.1 micron sterilizing grade filter or a series of such filters, can be used. After filtration, the serum is aseptically dispensed into bottles ensuring the sterility of the final product.
Using fertilized eggs as growth medium is an alternative to animal sera, used in large scale methods, for example in the production of for example of vaccines, and antibodies, for prophylactic, therapeutic and diagnostic purposes.
There are several manufacturers of animal sera for cell culture purposes, and all take great care to ensure that the products meet high requirements with regard to sterility, purity, and other quality criteria. One manufacturer lists the following properties, shown in Table 1 below:
TABLE 1Quality control of serum productsPhysical and chemical tests:Electrophoretic pattern, pH, osmolarity, total proteins, albumin, IgG,hemoglobin, globulinsBiochemical tests:Alanine transaminase (ALT), alkaline phosphatase, aspartaseaminotransferase (ast), bilirubin - total, bilirubin - direct,blood urea nitrogen (BUN), calcium, chloride, cholesterol, creatinine,creatinine kinase (CK), gamma-glutamyl transferase (GGT), glucose, highdensity lipoproteins (HDL), low density lipoproteins (LDL), inorganicphosphorous, potassium, sodium, triglycerides (TG), and uric acidMicrobiological tests:Sterility test (bacterial and fungal sterility tests according to currentUSP)Mycoplasma contamination (according to CFR)Viral contamination (according to CFR for bovine viral diarrhea (BVD),infectious bovine rhinotracheitis (IBR), and parainfluenza type 3 (PI3)Viral antibodies (determination of titer of antibodies to BVD, IBRand PI3)Endotoxins (Limulus amebocyte lysate (LAL) test using kineticturbidimetric method)
The manufacturers also test the stability and biological performance, i.e. checking the efficacy of the serum in promoting cell growth. However, as a precaution, manufacturers frequently underline that serum is a biological material, an undefined mixture of components in which the composition varies from one lot to another, and that some cell types are sensitive to variations in serum performance. As a consequence, customers are encouraged to evaluate serum samples using their own culture system and cells. In the meantime, the manufacturer reserves quantities of the specific lots until the testing is complete. In this way, a customer may choose the serum best suited for his particular applications (Product brochure E49/2 07/15 from Biological Industries Israel Beit Haemek Ltd., Kibbutz Beit Haemek, 25115, Israel).
Further, a large range of drugs and reagents used in therapy and diagnosis are currently synthesized using cell culture methods in large scale manufacturing facilities. Cell culture is also widely used in the vaccine industry, as well as in the production of antibodies. Lately, cell culture has also been used in the production of tissues and even organs, for use in reconstructive surgery and transplants. All these uses of serum and serum products involve extremely high requirements on reliability and repeatability.
The present inventor has recognized a need for improving the quality and reliability of cell culture media and cell culture methods. He has also set out to investigate the causative factors behind cancer, aiming at finding new approaches to prevent, alleviate or treat cancer.